Ancestral chromosomal signatures of Paenungulata (Afroteria) reveal the karyotype of Amazonian manatee (Trichechus inunguis, Sirenia: Trichechidae) as the oldest among American manatees

Background Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians. Results A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK). Conclusions TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis.

The order Sirenia are exclusively aquatic herbivorous mammals, composed of two families, Dugongidae (dugongs) and Trichechidae (manatees), that probably diverged in the early Eocene, 56 million years ago (myr) [6][7][8][9][10]. The Trichechidae family is divided into Miosireninae (extinct) and Trichechinae (current manatees) subfamilies. Three species of the Trichechus genus represent the current manatees, Trichechus manatus LINNAEUS 1758 (West Indian manatee), Trichechus senegalensis LINK 1795 (African manatee) and Trichechus inunguis NATTERER 1883 (Amazonian manatee). The taxon is distributed in the tropical and subtropical regions of the Atlantic Ocean: T. manatus lives in the Atlantic coastal region of the Americas, T. senegalensis in the rivers and coastal areas of western Africa and T. inunguis is endemic to Amazonian rivers [11].
Morphological data established the first phylogenetic relationships of trichequid representatives, suggesting that the first manatees have ancestry from estuarine regions and freshwater environments in South America [7,12,13]. Fossil analysis, through studies of tooth morphology, inferred that Ribodon limbatus AMEGHINO 1883 is an ancestor of the genus Trichechus [7,12,14]. Domning [7,12] proposed that T. inunguis is the most recent species among the representatives of Trichechus based on morphology and paleogeographic history.
The mitochondrial gene data described by Vianna et al. [15] strengthened the phylogenetic relationship between T. manatus and T. senegalensis, corroborating the morphological phylogenetic interpretations [7,12]. However, Cyt b genes in T. inunguis showed a lower degree of sequence changes concerning T. manatus and T. senegalensis, indicating the sequence in T. inunguis as the most conserved among Trichechus, although the study concluded that T. inunguis would be the most recent species. De Souza et al. [16] analyzed the mitochondrial genomes of Trichechus representatives and proposed the time of evolutionary divergence between the species at 6.5 myr. In addition, the study presented T. senegalensis as the oldest species among the Trichechus. It established a closer relationship between T. manatus and T. inunguis, mainly considering the divergence time at 1.34 myr between the two species. These divergence times are very short, considering the significant phenotypic differences between these species [11,16]. From a morphological perspective, it is possible to confirm the proximity between T. manatus and T. senegalensis due to the similarity in habitat and niches of these species, which contribute to the preservation of typical phenotypes in marine manatees. However, despite the genomic data by Vianna et al. [15] reinforcing this proximity of T. manatus and T. senegalensis, the findings in T. inunguis were controversial in relation to the phylogenetic interpretations already described for the species. The similarity of mitogenomes between T. manatus and T. inunguis described by De Souza et al. [16] proposes, for the first time, a different phylogenetic interpretation for the group.
Chromosome painting has been effective in clarifying information about evolutionary aspects of mammals and assessing karyotypic and phylogenetic ancestry, as well as evolutionary divergence between taxonomic groups [17,18]. Cytogenetic analyzes available in the literature for Trichechus showed the established diploid number (2n) and autosomal fundamental number (FN) for T. inunguis as 2n = 56/FN = 82 [19][20][21][22] and 2n = 48/ FN = 92 for T. manatus [22][23][24]. This variation in karyotypes is remarkable, with a difference of four Robertsonian rearrangements [19] between T. manatus and T. inunguis, considering the short divergence time (1.34 myr) between these species. More recent data from Noronha et al. [22] and De Oliveira et al. [20], based on karyotypic analysis, demonstrated chromosomes rearrangements and the natural occurrence of hybrids from reproduction between T. inunguis and T. manatus or different generations (F1, F2). Cytogenetic data for T. senegalensis [2,18,25]. Furthermore, Pardini et al. [2], using chromosome painting in T. m. latirostris (Sirenia), L. africana (Proboscidea), and P. capensis (Hyracoidea), established the karyotypic differences between these species and confirmed 11 synapomorphies that characterize the Paenungulata clade, in addition to establishing the ancestral karyotype (APK, 2n = 58). Therefore, the verification and number of chromosomal changes that have occurred during the divergence of T. manatus and T. inunguis could help to elucidate the phylogenetic interpretations described for the genus Trichechus. Here, data on chromosome painting in Trichechus inunguis, and the evolutionary aspects that differentiate the manatees T. manatus and T. inunguis and their phylogenetic relationships, are shown for the first time on a comparative chromosomal analysis with other representatives of the Paenungulata clade available from the published data.

Results
The karyotype of Trichechus inunguis (TIN) presents 2n = 56, FN = 92, and an XX/XY sex chromosome system. Of the autosome chromosomes, 19 pairs are biarmed and 8 one-armed; the X is submetacentric, and the Y is acrocentric.

Comparative analysis between TIN and TML
The comparative analysis between TIN and TML was proposed based on the results of Kellogg et al. [25], with hybridizations of Homo sapiens (HSA) probes in TML and the effects of hybridizations with TML probes in TIN of the present study. Therefore, the data found in TML were used as an intermediary to infer the chromosomal associations of HSA in TIN due to the high degree of genome similarity observed in the hybridizations between these species.  [2]. Paenungulata ancestral karyotype (APK) associations were also found in T. inunguis, with HSA 2/3 syntenies in two blocks (TIN 9 and TIN 12), 18/19 (TIN 7), 8/22 (TIN 14) (see Fig. 3 and Table 3). HSA 4/8 synteny is common in AEK and has been detected in Afroinsectiphilia (African insectivores) [26][27][28][29]. However, it was not observed in T. inunguis, as well as in L. africana, T. m. latirostris, and P. capensis [2,25,30], reinforcing that this association was lost in the representatives of Paenungulata.

The rapid dissemination of the Trichechus genus
The paleoenvironmental dynamics that occurred in South America during the Cenozoic were responsible for the diversification and distribution of the first representatives of the genus Trichechus [12]. During the formation of the Amazon basin, the Andean elevation generated different landscapes that benefited the diversity of the South American biota [31][32][33]. The discovery of the Potamosiren fossil links the first manatees to the estuarine and freshwater environments of South America [7,12]. The constant marine transgressions that occurred on the continent in the Neogene (Miocene and Pliocene) may have caused the reintroduction of sirenians into fresh waters, as the broad community of sirenians of the Tertiary was marine in origin [6,9,13,[32][33][34].
The first Trichechus diverged by allopatry in marine and freshwater environments. Within the Amazon basin, the Trichechus genus modified its diet; the high production of macrophytes and other abrasive grasses selected the first isolated Trichechus; outside the Amazon basin, marine Trichechus took different routes and diversified; Trichechus senegalensis, in coastal regions and rivers of tropical West Africa; and Trichechus manatus, in the coastal area of the American continents [12]. Fossil data for these manatees are still too scarce to suggest past distribution. However, the diversity of Trichechus manatus in the lineage-subspecies T. manatus bakerorum (extinct), T. manatus latirostris (Florida manatee), T. manatus manatus (Antillean manatee), and T. manatus manatus (Brazilian T. manatus) along the American Atlantic coast support a state of rapid   diversification within the genus Trichechus, validated by morphological, genomic and cytogenetic characteristics [14,15,[35][36][37][38].
Although phylogenetic positions are still controversial among extant Trichechus [12,15,16,39], genomic data have estimated the time of evolutionary divergence between these species. The analysis by Cantanhede et al. [36] with D-loop between T. manatus and T. inunguis estimated the time of evolutionary divergence from 3.1 to 0.65 myr, while the complete mitochondrial genomes analyzed by De Souza et al. [16] showed an evolutionary divergence between T. manatus and T. inunguis of 1.34 myr. The short time of divergence between these species can be seen in our data due to the high chromosomic similarity found in the present study, which can also support the existence of natural hybridization between T. manatus and T. inunguis in the Amazon estuary [20,22]. The estimated rate of chromosomal changes in Paenungulata is considered slow to moderate (0.09 -0.16 changes per 1 million years -changes/myr) compared to other mammalian groups [2]. The chromosomal changes for the paenungulate of the orders Hyracoidea (P. capensis -2n = 54) and Proboscidea (L. africana -2n = 56) show a difference of 6 to 9 changes in APK, respectively, given that the evolutionary divergence of these taxa has been approximately 56 myr [1,40]. In addition, other known representatives of Hyracoidea (Dendrohyrax arboreus, 2n = 54; Heterohyrax hrucei: 2n = 54) and Proboscidea (Elephas maximus, 2n = 56) still maintain a conserved diploid number [41,42]. However, the difference of four Robertsonian translocations and a pericentric inversion between T. inunguis (2n = 56) and T. manatus (2n = 48) reveals a high rate of chromosomal changes within the genus Trichechus, between 1 to 5 changes/myr. The analysis of the Cyt b gene by Vianna et al. [15] suggested that T. inunguis might belong to an older lineage of manatees adapted to freshwater. Therefore, the species may have a more conserved gene sequence than T. manatus and T. senegalensis. The new insights of De Souza et al. [16] on the phylogenetic relationship of T. manatus and T. inunguis provide more specific answers about the differences between these species, which were also reinforced in the present study. The chromosomal changes in APK that led to the karyotype of T. manatus and T. inunguis range from 7 to 4 changes, respectively; this indicates that T. inunguis shares a more conserved karyotype with APK, while T. manatus presents apomorphies that show a condition that is more derived from APK. Notably, the chromosomal evolution of the Trichechus genus will be elucidated only after the application of TML probes to T. senegalensis.

Conclusion
Here, we evaluated by chromosome painting important data on the karyotypic differences between the species Trichechus manatus and Trichechus inunguis and the phylogenetic relationships of these species to other representatives of Paenungulata. The high rate of chromosomal changes in manatees shows them as outliers of the Afrotheria clade. Despite this, the homeologies between the paenungulate karyotypes are still very conserved, with evidence even in the G-banding pattern. The shared HSA syntenies in T. inunguis reveal it as a representative of the placental mammalian taxons Afrotheria and Paenungulata. The phylogenetic signals found in T. inunguis show that the species shares more conserved chromosomal signals with the ancestral karyotype of Paenungulata (APK) compared to hyrax (Procavia capensis), the African elephant (Loxodonta africana), and Florida manatee (Trichechus manatus latirostris). From a phylogenetic perspective, the karyotype of T. m. latirostris is the most derived among the representatives of Paenungulata. Furthermore, the data from this study also point to the phylogenetic position between T. manatus and T. inunguis, showing that T. manatus presents a more recent condition than T. inunguis among the American Trichechus. However, complete understanding of the chromosomal evolution of the genus will be possible only after chromosomal painting of T. senegalensis.

Methods
Blood samples were collected from a male and a female of Trichechus inunguis under the SISBIO license number (Number: 44915-1). Chromosomal preparations were obtained from temporary lymphocyte cultures. Cultivation was performed in RPMI 1640 medium (Vitrocell) with fetal bovine serum (FBS) and phytohemagglutinin and incubated at 37ºC in 5% CO 2 for 96 h. Metaphases were analyzed according to chromosome morphology and organized karyotype according to Assis et al. [19]. The G-banding pattern was performed using Seabright's protocols [43], the best G banded karyotype was published for us in Noronha et al. [22]. The whole chromosome probes used in this study were described by Pardini et al. [2], where 23 peaks were generated from a male of Trichechus manatus latirostris (TML; 2n = 48) by flowsorted, with 17 peaks of a single chromosome (TML 1,5,8,9,10,11,12,14,15,16,17,18,20,20,22,23, Y) and 3 peaks composed of two chromosomes (2 + 4, 3 + 7 and 6 + X). The TML 20 chromosome is present in 2 separate peaks, possibly due to the heterochromatin difference between homologs carrying the nucleolus organizer region (NOR) and presenting nonspecific markings on the chromosomes. TML chromosomes 11, 14, and 17 have both peaks in their pure form and also mixed peaks with other chromosomes, such as 11 + 13, 14 + 19, and 17 + 21, making it possible to characterize the TML chromosome 19 in hybridizations (Table 1).
In situ hybridizations were performed according to Yang and Graphodatsky [44], photographed with a Zeiss Axiocam camera, coupled to a Zeiss microscope, and analyzed with AxioVision Rel software. 4.6. The analyzes followed the interpretation of the presence/absence of signals in the chromosomes; comparative idiograms were set up in Photoshop CS6 software for cytogenetic analysis between the investigated species.